Abstract Substance use disorders and subsequent relapse occurs as a result of long-term or permanent changes in the neurocircuitry of motivated behavior (drug-induced neuroplasticity). My recent studies have uncovered a discrete region of the lateral hypothalamus (LHA) that critically contributes to cocaine-induced behaviors, but its role in drug addiction was unknown until now. This region is striking in that it is heavily wrapped in a specialized extracellular matrix structures called perineuronal nets (PNNs). PNNs are an emerging and promising target for preventing drug-induced neuroplasticity because their removal profoundly alters neuroplasticity by cocaine. My recent studies have found and defined a discrete region of the dorsal and intermediate zones of the anterior LHA (LHAad/i) with remarkably robust expression of PNNs around GABAergic neurons that I have discovered to be critical for cocaine- but not sucrose-induced behaviors. The LHAad/i receives input from the prelimbic prefrontal cortex (PL PFC) and sends projections to the VTA. PL PFC and the VTA are major components of the mesocorticolimbic circuitry of motivated behavior and intimately involved in relapse of drug seeking. The role of the LHAad/i within this circuitry has not been studied but is importantly involved in cocaine-seeking behavior. Notably, it is not a part of the more heavily studied regions of the lateral hypothalamic area. I have established that these PNNs are critical for the acquisition of cocaine- but not sucrose-induced behaviors and for cue-induced reinstatement of cocaine seeking. Here I propose to test three hypotheses in three Specific aims that focus on the mechanism and circuitry underlying the prevention of cocaine-induced behavior by LHAad/i PNN removal. Specific Aim 1 (K99 period) will test the hypothesis that LHAad/i PNN removal will increase presynaptic GABA input on VTA DA neurons. I will be formally trained in ex vivo electrophysiology and optically stimulate LHAad/i terminals within the VTA to define the impact of LHAad/i PNN removal on synaptic transmission of VTA neurons. I will begin Specific Aim 2 during the last part of the K99 phase. Specific Aim 2 (K99 and R00 period) will test the hypothesis that chemogenetic activation of LHAad/i neurons that project to the VTA will block cue-induced reinstatement of cocaine seeking. I will continue my training in the use of advanced viral vectors to chemogenetically activate LHAad/i neurons that project to the VTA. Specific Aim 3 (R00 period) will test the hypothesis that LHAad/i PNN removal will reduce PL PFC glutamate input onto LHAad/i neurons. PL PFC projections to the LHAad/i will be optogenetically stimulated. This Aim will be conducted independently during years 4 and 5 of the R00 period to apply the training I received during the K99 period. Central to successful long-term relapse prevention is the need to modify new targets that underlie cocaine-induced plasticity within the circuitry of motivated behavior. The proposed training is fundamental for the PI?s successful transition from postdoctoral fellow to an independent tenure-track faculty position.